Efficient Cell and Cell-Sheet Harvesting Based on Smart Surfaces Coated with a Multifunctional and Self-Organizing Elastin-Like Recombinamer María Pierna, Mercedes Santos, Francisco J. Arias, Matilde Alonso, and Jose ́ C. Rodríguez-Cabello* Bioforge Group, University of Valladolid, CIBER-BBN Paseo de Bele ́ n 11, 47011 Valladolid, Spain * S Supporting Information ABSTRACT: A wide range of smart surfaces with novel properties relevant for biomedical applications have been developed recently. Herein we focus on thermoresponsive surfaces that switch between cell-adherent and nonadherent states and their applications for cell harvesting. These smart surfaces are obtained by covalently coupling a tailored elastin- like recombinamer onto glass surfaces by means of the well- known and widely applied Click Chemistry methodology. The resulting recombinamer-functionalized surfaces have been characterized by means of water contact angle measurements, XPS and TOF-SIMS. A cell-based analysis of these surfaces with human fibroblasts showed a high degree of adhesion to the surface in its adherent state (37 °C), thus, promoting cell viability and proliferation. A temperature decrease triggers reorganization of the recombinamer, thus, markedly increasing the number of nonadherent domains and masking the adherent ones. This process allows a specific and efficient temporal control of cell adhesion and cell detachment. After determination of the properties required for a suitable cell-harvesting system, optimization of the process allows single cells or cell sheets from at least two types of cells (HFF-1 and ADSCs) to be rapidly harvested. ■ INTRODUCTION Cell-harvesting systems and technologies are key elements in the development of cell production techniques, especially as regards their subsequent use in human therapies. These systems must be considered as relevant enabling technologies since areas such as tissue engineering and regenerative medicine will not easily become universally applicable without a reliable source of cells for therapeutic purposes. Most cell lineages need to adhere to a substrate to proliferate. As a result, most of the protocols currently used to amplify cell numbers require the use of cell-adherent supports. However, this poses a technical challenge in classical approaches as the cells subsequently need to be harvested from the substrates prior to use. Classical solutions to this problem tend to be based on two different approaches, namely, mechanical and proteolytic enzyme harvesting. 1 However, both of these techniques may compromise cell viability and are mainly restricted to flat substrates. Although trypsinization is currently the most widely used proteolytic method, it has some major drawbacks. The most obvious of these is the previously cited cell viability, as excessive exposure of the cultured cells to trypsin activity may damage many different membrane proteins and compromise cell survival. 2 Likewise, trypsinization protocols are time-consuming and are strongly dependent on the operator’s expertise. Moreover, the utilization of trypsin, especially in the industrial production of cells for human use, raises the problem of trypsin elimination from the final product. In other words, the manufacturer must be able to conclusively prove that no trypsin remains in the final product. Additionally, for those cell cultures involving cell differentiation, trypsiniza- tion destroys most of the cellular markers that are currently used to determine the resulting cell lineage. 3 Recent studies by Okano and co-workers have opened up a new possibility in this field, namely, the use of a smart surface that can switch between a cell-adherent and nonadherent state as a result of a change in temperature. 4 In such an approach, poly(N-isopropylacrylamide) (PIPAAm) and its derivatives are grafted onto an appropriate substrate and builds a brush-like structure of the responsive material directly on the surface. In this case, changes in the apparent surface hydrophobicity and, therefore, the physical properties of the surface, seem to govern the mechanism that allows such systems to work. Based on the well-known lower critical solution temperature (LCST) behavior of PIPAAm, the polymer switches between a hydrophobic state at temperatures above its LCST and a hydrophilic state below it, as does the surface on which PIPAAm was grafted. This approach has led to significant progress in this field providing harvesting protocols required are neither complex nor time-consuming and there are no concerns as regards contamination from the reagents used during the harvesting protocol. 5 In addition, and more importantly, this approach means that both cells and cell Received: February 20, 2013 Revised: April 24, 2013 Article pubs.acs.org/Biomac © XXXX American Chemical Society A dx.doi.org/10.1021/bm400268v | Biomacromolecules XXXX, XXX, XXX-XXX